The biological behavior and clinical outcome of pituitary adenoma are affected by the microenvironment

Abstract Background Pituitary adenoma is one of the most common brain tumors. Most pituitary adenomas are benign and can be cured by surgery and/or medication. However, some pituitary adenomas show aggressive growth with a fast growth rate and are resistant to conventional treatments such as surgery, drug therapy, and radiation therapy. These tumors, referred to as refractory pituitary adenomas, often relapse or regrow in the early postoperative period. The tumor microenvironment (TME) has recently been identified as an important factor affecting the biological manifestations of tumors and acts as the main battlefield between the tumor and the host immune system. Main body In this review, we focus on describing TME in pituitary adenomas and refractory pituitary adenomas. Research on the immune microenvironment of pituitary adenomas is currently focused on immune cells such as macrophages and lymphocytes, and extensive research and experimental verifications are still required regarding other components of the TME. In particular, studies are needed to determine the role of the TME in the specific biological behaviors of refractory pituitary adenomas, such as high invasion, fast recurrence rate, and high tolerance to traditional treatments and to identify the mechanisms involved. Conclusion Overall, we summarize the similarities and differences between the TME of pituitary adenomas and refractory pituitary adenomas as well as the changes in the biological behavior of pituitary adenomas that may be caused by the microenvironment. These changes greatly affect the outcome of patients.


| INTRODUC TI ON
3][4][5][6] The clinical consequences of refractory pituitary adenoma are serious, such as tumor growth, cranial nerve compression, bone destruction and invasion, and excessive hormone secretion, which seriously affect the health of patients.
][9][10] Current research on the TME has mainly focused on the immune cells, especially tumor-related macrophages 11,12 ; however, nonimmune cells and noncellular components also play important roles in the immune microenvironment. 13There are many similarities and differences in the immune microenvironment between refractory pituitary adenoma and common pituitary adenoma.These factors affect the occurrence, development, and prognosis of pituitary adenoma and refractory pituitary adenoma and determine the treatment strategy of different types of pituitary adenoma to a certain extent.
Refractory pituitary adenoma and common pituitary adenoma have many similarities and differences in the tumor immune microenvironment, which contribute to the special biological characteristics of refractory pituitary adenoma, such as strong invasion ability, early postoperative recurrence, stronger drug resistance, and so on.This review therefore considers the immune microenvironment and then explores the reasons for the invasiveness, drug resistance, and postoperative recurrence of pituitary adenomas, especially refractory pituitary adenomas, and the factors affecting the immune microenvironment of pituitary adenomas.

| DEFINITI ON OF REFR AC TORY PITUITARY ADENOMA
According to the 2017 WHO Classification of pituitary adenomas and the Expert Consensus on Diagnosis and Treatment of refractory pituitary adenomas in China (2019), the definition of refractory pituitary adenomas is summarized as follows: Refractory pituitary adenoma shows aggressive growth on imaging, and its growth rate is faster than that of general pituitary adenoma.It is resistant to conventional treatment, such as surgery, drug therapy, and radiation therapy, and often relapses or regrows in the early postoperative period. 3,5The 2017 WHO Classification of pituitary adenomas emphasizes that refractory pituitary adenomas have the following five characteristics: I. Invasive growth.II.Rapid growth.III.After multimodality standardized treatment such as surgery, drugs, and radiation therapy, tumors continue to grow and/or secrete excessive hormones.IV.Tumor enlargement and/or high levels of hormone secretion seriously affect patients' quality of life and threaten their lives.V.The prognosis of the patient is poor, but intracranial and distant metastasis of the tumor are not present 5 (Table 1).
Most refractory pituitary adenomas are aggressive.Invasiveness is also an important biological feature and a key factor in the poor prognosis of refractory pituitary adenomas.In addition, invasive pituitary adenoma has a higher risk of tumor progression, which is one of the main reasons why surgical resection is difficult.It is worth mentioning that not all invasive pituitary adenomas are refractory pituitary adenomas, but tumor invasiveness is indeed one of the main clinical features of refractory pituitary adenomas.Therefore, in reviewing the effects of TME on the biological behavior of refractory pituitary adenomas and the differences between refractory and common pituitary adenomas, we will focus on the factors related to tumor aggressiveness.

| CHANG E S IN THE TUMOR IMMUNE MI CROENVIRONMENT MAY C AUS E PITU ITARY ADENOMA S TO DE VELOP A MORE " MALIG NANT " PHENOT YPE
Refractory pituitary adenoma is defined as a tumor with aggressive growth on imaging.However, the definition of refractory pituitary adenoma is not clear, and there is no specific pathological classification at present. 14,1517 Refractory pituitary adenomas are reported to occur in about 10%-45% of clinically detected pituitary adenomas.According to statistics, about 0.5% of pituitary adenomas have an invasive course.5][26] It is therefore important to understand the immune microenvironment of refractory pituitary adenomas.

| Macrophage
Macrophages are usually the most abundant group of immune cells in the TME, 11,28,29 including in pituitary adenomas.1][32] Two studies have successively confirmed the presence of M1 and M2 macrophages in estrogeninduced prolactinoma pituitary (Figure 1. Procedure ⑤) and nonfunctioning pituitary adenomas. 33,34In addition, single-cell transcriptome and single-cell multiomics analyses revealed that macrophages and T lymphocytes were abundant in pituitary adenomas (most of the cells were still tumor cells).Immunohistochemical staining also confirmed the validity of the single-cell sequencing results. 35In addition, the ratio of the M2 marker CD163 to the M1 marker HLA-DR was about three times higher in pituitary adenomas than in normal pituitary tissue, indicating a higher proportion of M2-type macrophages in pituitary adenomas. 30In addition, levels of the M2 polarization factor interleukin (IL)-4 have been reported to be about five times higher in pituitary adenomas compared with levels of the M1-type factor interferon (IFN)γ, and the ratio corresponds to the M2/M1 cell ratio 30 (Figure 1, Procedure ②).Other studies reached similar conclusions. 36,37[40][41] In turn, pituitary adenomas may also have a special pathway to stimulate the migration of macrophages to the region where the tumor cells are located, which further affects the composition of the immune microenvironment within the tumor 31 (Figure 1, Procedure ①).According to the above results, it can be inferred that M2 macrophages may also play a role in the occurrence and development of pituitary adenomas. 33gnificantly enhanced invasion ability is one of the important characteristics of refractory pituitary adenoma, and macrophages can affect the invasion ability of pituitary adenoma in many ways.
AIP mutation-positive pituitary adenomas include large numbers of infiltrating macrophages, and further studies showed that the role of normal AIP expression in maintaining the normal state of pituitary adenomas appears to be important, as positive or low AIP mutation expression is indicative of a more aggressive pituitary adenoma phenotype 31,42 (Table 2).Further studies have explored the specific mechanism by which macrophages affect the invasion ability of pituitary adenoma: on one hand, pituitary adenomas cause acidification of the TME, resulting in polarization of tumor-related macrophages into an M2-like phenotype (Figure 1, Procedure ③), whereas on the other hand, M2-like macrophages secrete CCL17 and enhance the invasion ability of pituitary adenoma cells via the CCL17/CCR4/mammalian target of rapamycin complex 1 axis 43 (Figure 1, Procedure ④).Other studies have shown similar results.In a subtype of nonfunctional pituitary adenoma, macrophages were recruited and polarized into an M2-like phenotype, a process that increased the invasion ability of this subtype of nonfunctional pituitary adenoma and the proliferation of tumor cells. 34However, functional pituitary adenomas and gonadotropin tumors can also recruit macrophages and subsequently convert them into M2-like phenotypes, and an analysis of clinical data confirmed that the percentage of M2-like macrophages was significantly correlated with the aggressiveness of gonadotropin pituitary adenomas. 37Not only that, other studies have suggested a positive correlation between the degree of infiltration of macrophages and the size of pituitary adenomas, with larger adenomas containing more macrophages. 32 summary, macrophages may affect the function of pituitary adenoma cells in various ways, and ultimately lead to the enhancement of invasion and proliferation ability of refractory pituitary adenoma.However, there are still no studies to investigate the relationship between refractory pituitary adenoma and macrophages in the immune microenvironment.However, according to the above, we hypothesized that the polarization and enrichment of macrophages may be closely related to the formation of refractory pituitary adenoma.

| T lymphocyte
T cells play a key role in tumor immunity, and the degree of their invasion in tumors is closely related to tumor prognosis. 44,45Recent studies highlighted that regulatory T lymphocytes and cytotoxic T lymphocytes are the major T cell types with play a very important and almost opposite role in tumor immunity.Therefore, in the following review, T lymphocytes in the pituitary adenoma microenvironment were discussed around these two cell groups.

| Regulatory T lymphocytes
By suppressing antitumor immunity, regulatory T cells (Tregs) can prevent protective immunity from monitoring tumors.Thus, the body loses the effective antitumor immune response.Eventually, this process will promote the development of tumors. 46,47In most cancers, a high ratio of Tregs/CD8 + T cells usually indicates a poor prognosis. 48In pituitary adenomas, studies have found that more aggressive and poor prognostic adenomas tend to have significant upregulation of Treg cells. 49,50I G U R E 1 Macrophages in the immune microenvironment of pituitary adenoma: Procedure ①: Macrophage recruitment is promoted through the CCL5-CCR5 pathway, which mainly occurs in AIP mutation-positive pituitary adenomas 31 ; Procedures ②, ③, ⑤: IL-4, lactic acid, and DES can induce macrophages to polarize into M2 phenotype 30,33,43 ; Procedure ④: M2-type macrophages can improve the invasion ability of tumor cells through the CCL17-CCR4 pathway. 63

| Cytotoxic T lymphocytes
In general, cytotoxic T cells target host cells infected by viruses or cancerous cells.They induce apoptosis of target cells by secreting perforin and granzyme.However, the effect of cytotoxic T lymphocytes on pituitary adenoma is still controversial.For example, silent type III pituitary adenomas with less cytotoxic T lymphocyte infiltration have a higher risk of recurrence and invasion. 51Another study of growth hormone-secreting pituitary adenomas showed that the accumulation of cytotoxic T lymphocytes (CD8 + T cells) in the pituitary adenoma microenvironment was negatively correlated with invasive behavior and drug resistance.This result is consistent with the effect of cytotoxic T cells on pan carcinoma. 52wever, a case report of an aggressive hormone-secreting pituitary adenoma showed high levels of CD8+ lymphocyte infiltration in tumor tissue.The author suggested that the increase in tumor invasiveness and drug resistance in this case may be related to the infiltration of CD8+ lymphocytes 53 (Figure 2A).
In addition, refractory pituitary adenomas also have something in common with nonrefractory pituitary adenomas.In one study, the Th1/Th2 ratio was significantly reduced after surgery, indicating cross-regulation among Th cell subsets, and playing an important role in the inflammatory lesions of pituitary adenoma tissue 54 (Figure 2B).
In conclusion, the presence and proportion of regulatory T lymphocytes and cytotoxic T lymphocytes in the pituitary adenoma microenvironment are largely related to the invasion ability, drug resistance, and other biological characteristics of pituitary adenoma.
Therefore, it is very important to conduct in-depth studies on each subgroup of T lymphocytes in the refractory pituitary adenoma microenvironment.It may profoundly affect the future immune diagnosis and treatment of refractory pituitary adenoma.

| Tumor-associated fibroblast
TAFs are the most abundant population in the tumor stroma of refractory pituitary adenomas.Invasive pituitary adenoma-derived TAFs have been shown to significantly promote the growth of rat pituitary cells both in vitro and in vivo, and VEGF was overexpressed in both invasive pituitary adenoma-derived TAFs and tumor specimens. 55In addition, the proliferation, migration, and invasion of human pituitary adenoma cells were inhibited by small-interfering RNA-mediating fibroblast growth factor gene silencing, further confirming the role of TAF and fibroblast growth factor in various biological behaviors of refractory pituitary tumors. 56Although there are few studies on TAF in pituitary adenoma at present, it can be found that TAF is an important part of the pituitary adenoma microenvironment through the above research reports.Therefore, future studies are still needed to further analyze the relationship between TAF and malignant phenotype of refractory pituitary adenoma.

| Extracellular matrix
The ECM plays an important role in tumor invasion and metastasis and may be related to the formation of refractory pituitary adenomas. 57The function of the ECM is affected by the dynamic balance between MMPs (matrix metalloproteinase) and TIMPs (tissue inhibitor of matrix metalloproteinases), and a systematic review and meta-analysis involving 1320 patients with pituitary adenomas found elevated expression levels of MMP-9 and MMP-2 in patients with invasive pituitary adenomas, but no difference in TIMPs. 57Further studies are needed to confirm these results.
PD-L1 transcription levels were significantly higher in primary pituitary adenomas than in recurrent pituitary adenomas. 58In addition, PD-1/PD-L1 was also associated with the invasiveness of pituitary adenomas.In one study, the expression of PD-L1 was higher in invasive pituitary adenomas than in pituitary adenomas without invasive behavior. 59Therefore, more studies are needed on the relationship and mechanism between PD-1/PD-L1 axis and immune escape of pituitary adenoma.

| Cytokines
Cytokines play a key role in various physiological and pathological activities of cells.Epidermal growth factor (EGF) and its receptor (EGFR) are prevalent in mammalian pituitary adenomas, including in humans, mice, and dogs 60 (Figure 3A), and are expressed in different ways in all categories of human pituitary adenomas. 61,62scular endothelial growth factor (VEGF) plays a key role in immunosuppressive microenvironments by inhibiting dendritic cell maturation. 63Studies have confirmed that VEGF-A is not only expressed in vascular endothelial cells, but also in tumor cells in pituitary adenomas (Figure 3B), while VEGF-A/VEGF receptor (VEGFR) 1 expression was significantly upregulated in nonfunctional pituitary adenomas invading the cavernous sinus (Figure 3C), suggesting a positive role of VEGF and its receptors in the development and invasion of pituitary adenomas. 59,64In addition, the TAF-related cytokines IL-6 and CCL2 mentioned above were also positively correlated with the aggressiveness and proliferation index of pituitary adenoma, respectively 65 (Figure 3D,E).
In summary, IL-6, CCL2, EGF/EGFR, VEGF/VEGFR, and other cytokines are expressed in pituitary adenomas and are generally related to their aggressiveness, occurrence, and development.
Immunotherapy targeting these cytokines may thus represent an important future direction of immune checkpoint therapy for pituitary adenomas.

F I G U R E 2 Effect of T lymphocytes on immune microenvironment of pituitary adenoma. (A) Regulatory T cells and cytotoxic T cells may enhance the invasion ability of pituitary adenoma;
(B) Preoperative and postoperative changes of TH cell-associated cytokines in pituitary adenomas.

| Cytoskeleton
Cytoskeleton is a complex multifunctional network of dynamic protein filaments.It plays an important role in maintaining the cell shape and structure, and in cell movement, differentiation, and intracellular transport. 66A recent study confirmed that coexpression of intermediate filament glial fibrillary acidic protein and cytokeratin in pituitary adenomas and pituitary cells was associated with hormone expression and low recurrence rates. 67e recurrence rate is also one of the important differences between refractory pituitary adenomas and nonrefractory pituitary adenomas.

| Neuropeptides
Downregulation of GAL and GAL receptors was found in some pituitary adenoma tumor tissue samples, and expression levels in different subtypes of pituitary adenomas were negatively correlated with tumor proliferation. 68GAL-positive subtypes tended to have higher cure rates, in accordance with similar results observed for GAL and its receptors in gliomas. 68

| Hormones and their receptors
Abnormal hormone secretion is one of the important signs of refractory pituitary adenoma.A study found that expression levels of FSH receptors were significantly higher in invasive pituitary adenomas (68%) compared with noninvasive pituitary adenomas (12%), suggesting that FSH receptors may be a marker of aggressiveness in pituitary adenomas. 69e differences and similarities between refractory pituitary adenoma and pituitary adenoma tumor immune microenvironment at the cellular and molecular levels are summarized in Table 2.

| B I OLOG I C AL B EHAVI OR S REL ATED TO THE IMMUNE MICROENVIRONMENT
The immune microenvironment is closely related to the size, infiltration ability, hormone-secretion ability, and other specific biological behaviors of pituitary adenomas 12,70 (Table 3).

| Bone invasion
Bone invasion and destruction are typical biological behaviors of invasive pituitary adenomas, with bone-invasive pituitary adenomas accounting for 6.4% of all pituitary adenomas. 713][74][75][76] Some authors thus believe that cathepsin K may also be indispensable for the bone-destruction behavior of pituitary adenomas. 77However, further studies are needed to clarify this issue.
Regarding the mechanism of bone damage, Zhu et al. reported that MEG8 promotes TNFα expression by sponging miR-454-3p, which ultimately leads to the occurrence of bone destruction, and was associated with poor progression-free survival. 78Another study explored the mechanism of bone invasion in pituitary adenoma tumor cells in vitro and found that GT1.1 cells activated the phosphoinositide 3-kinase-Akt, mitogen-activated protein kinase, and calcium signaling pathways through the RASSF10 (Ras Association Domain Family Member 10)-MDM2 (MDM2 Proto-Oncogene) pathway, and promoted the differentiation of RAW264.7 cells into osteoclasts through exosomes, resulting in bone destruction. 79Zhang et al. found that bone destruction required the involvement of osteopontin secreted by fibroblasts, which may lead to bone destruction in patients with NFPA by altering the expression and polymerization of caldesmon in fibroblasts. 80

| Angiogenesis
The continuous division and proliferation of tumor cells require a large amount of oxygen and nutrients, which rapidly leads to the impoverishment and anoxia of the microenvironment.In order to survive under unfavorable conditions, tumor cells thus need to adapt and adjust to the environment, to develop into a more malignant state.During this process, blood vessel-like formation occurs within the tumor tissue. 81Numerous studies have investigated the hypoxic environment and key factors such as angiogenesis in gliomas. 82Human pituitary adenomas are generally considered to demonstrate more active angiogenesis than normal healthy pituitary tissue, 83 and angiogenesis in pituitary tumors is mainly regulated by PGF (Placental Growth Factor) and VEGF. 84Studies have demonstrated that epidermal growth factor-like domain 7 (EGFL7) is widely involved in GHPAs (hormone-producing pituitary adenomas) angiogenesis, tumor growth, invasion, and metastasis and inhibits GHPAs growth and invasion by decreasing the expression of this molecule. 62,85 addition, vasculogenic mimicry occurs in pituitary adenomas, which refers to the phenomenon in which vascular-like channels, which are not lined by endothelial cells, are formed in tumors.
Vasculogenic mimicry is seen in both recurrent and nonrecurrent nonfunctional pituitary adenomas, but not in normal pituitary tissues. 86

| Recurrence and invasion
Silent type III pituitary adenomas are more dangerous than conventional acellular adenomas in terms of the risks of invasion and recurrence. 51Whole-transcriptome analysis showed that secreted proteins that inhibited T lymphocyte activity were upregulated in SS-3 adenomas, while proteins associated with cytotoxic T lymphocyte antigen presentation were downregulated and the overall concentration of T lymphocytes was relatively reduced. 51udies have confirmed that EFGR was overexpressed in recurrent growth hormone-secreting pituitary adenomas and invasive silent subtype III adenomas, suggesting that EGF/EGFR expression may be related to the recurrence and aggressiveness of pituitary adenomas. 61Similar results were obtained in another study, which showed higher expression levels of EGFL7 and P-EGFR in invasive  and promoting VEGFA expression. 87By comparing the expression levels of the above three factors in pituitary tissues, He et al. found that the expression levels of RSUME and hypoxic-inducing factor-1α receptor were significantly higher in invasive pituitary adenomas compared with noninvasive pituitary adenomas. 87e similarities and differences in the biological behavior of pituitary adenomas and refractory pituitary adenomas, and the relationship between these phenomena and the TME are summarized in Table 3.

| CURRENT S TATUS OF IMMUNOTHER APY FOR PITUITARY ADENOMA
Surgical treatment is currently the first choice of treatment for pituitary adenomas and refractory pituitary adenomas; however, the postoperative recurrence rate of some tumors remains high. 18,88,89 addition to surgery and radiation therapy, temozolomide is currently the only oral chemotherapeutic drug considered to be effective for pituitary adenomas, and some studies have suggested that early treatment with temozolomide may also have positive effects in patients with functional invasive pituitary adenomas. 17,90munotherapy is a promising treatment that has shown clinical activity in many cancer types. 91,92Previous studies have explored pituitary adenomas and invasive pituitary adenomas at the molecular level, and have summarized the therapeutic targets and clinical experience. 93Many studies [94][95][96] have shown that exosomes regulated the TME and were closely related to tumor immunotherapy, and corresponding studies and clinical trials have been conducted in patients with gliomas. 97These results thus provided a promising direction for further exploration of immunotherapy for pituitary adenomas.
Lin et al. treated a female patient with an invasive ACTH-pituitary adenoma with the combination of ipilimumab and nivolumab with good therapeutic effect and suggested that this combination might provide an effective treatment for pituitary cancer. 98 addition, DCs (dendritic cells) in invasive pituitary adenomas were shown to be activated by mucin 1 and polyinosinic:polycytidylic acid (poly I:C). 99Mucin 1 is an epithelial membrane-associated glycoprotein and an important molecular marker, with obvious positive expression in invasive pituitary adenoma, while poly I:C is often used as an immune stimulant or adjuvant.In this study, mucin 1 + poly I:C stimulated the expression of co-stimulatory molecules to polarize the immune response toward Th1, and ultimately triggered the progression of antitumor immunosuppressive pituitary tumors. 99e above summary indicated a lack of clinical applications relating to current immunotherapy research on pituitary adenomas, and there is currently no systematic treatment direction or strategy.
Notably, however, attitudes toward immunotherapy remain positive, and further studies are needed to identify treatments that take account of the specific immune status of pituitary adenomas and refractory pituitary adenomas.

| CON CLUS IONS
In conclusion, the immune microenvironments of pituitary adenomas and refractory pituitary adenomas are dynamic and complex, and influence the whole process of diagnosis, treatment, and prognosis of pituitary adenomas.Although the immune microenvironment and immunotherapy represent a promising direction for future exploration and research in relation to pituitary adenomas, relevant research is currently lacking and further studies are needed to explore this issue.

F I G U R E 3
Effect of cytokines on immune microenvironment of pituitary adenoma.(A) Epidermal growth factor (EGF) and its receptor (EGFR) are prevalent in mammalian pituitary adenomas, including in humans, mice, and dogs.(B) VEGF-A is present in vascular endothelial cells and pituitary adenoma tumor cells.(C, D) VEGFR-1 and IL-6 may be related to the invasive behavior of pituitary adenoma.(E) CCL-2 may be related to the proliferative behavior of pituitary adenoma.TA B L E 3 Distinction between refractory pituitary adenoma and pituitary adenoma (2).